ResearchPad - fungal-genetics https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Short-term evolution strategies for host adaptation and drug escape in human fungal pathogens]]> https://www.researchpad.co/article/elastic_article_14537 <![CDATA[Quantitative real-time PCR as a promising tool for the detection and quantification of leaf-associated fungal species – A proof-of-concept using Alatospora pulchella]]> https://www.researchpad.co/article/5989db52ab0ee8fa60bdc5cf

Traditional methods to identify aquatic hyphomycetes rely on the morphology of released conidia, which can lead to misidentifications or underestimates of species richness due to convergent morphological evolution and the presence of non-sporulating mycelia. Molecular methods allow fungal identification irrespective of the presence of conidia or their morphology. As a proof-of-concept, we established a quantitative real-time polymerase chain reaction (qPCR) assay to accurately quantify the amount of DNA as a proxy for the biomass of an aquatic hyphomycete species (Alatospora pulchella). Our study showed discrimination even among genetically closely-related species, with a high sensitivity and a reliable quantification down to 9.9 fg DNA (3 PCR forming units; LoD) and 155.0 fg DNA (47 PCR forming units; LoQ), respectively. The assay’s specificity was validated for environmental samples that harboured diverse microbial communities and likely contained PCR-inhibiting substances. This makes qPCR a promising tool to gain deeper insights into the ecological roles of aquatic hyphomycetes and other microorganisms.

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<![CDATA[Unusual genome expansion and transcription suppression in ectomycorrhizal Tricholoma matsutake by insertions of transposable elements]]> https://www.researchpad.co/article/Nd7412b83-0508-48a9-959e-b3aa8ede7a25

Genome sequencing of Tricholoma matsutake revealed its unusually large size as 189.0 Mbp, which is a consequence of extraordinarily high transposable element (TE) content. We identified that 702 genes were surrounded by TEs, and 83.2% of these genes were not transcribed at any developmental stage. This observation indicated that the insertion of TEs alters the transcription of the genes neighboring these TEs. Repeat-induced point mutation, such as C to T hypermutation with a bias over “CpG” dinucleotides, was also recognized in this genome, representing a typical defense mechanism against TEs during evolution. Many transcription factor genes were activated in both the primordia and fruiting body stages, which indicates that many regulatory processes are shared during the developmental stages. Small secreted protein genes (<300 aa) were dominantly transcribed in the hyphae, where symbiotic interactions occur with the hosts. Comparative analysis with 37 Agaricomycetes genomes revealed that IstB-like domains (PF01695) were conserved across taxonomically diverse mycorrhizal genomes, where the T. matsutake genome contained four copies of this domain. Three of the IstB-like genes were overexpressed in the hyphae. Similar to other ectomycorrhizal genomes, the CAZyme gene set was reduced in T. matsutake, including losses in the glycoside hydrolase genes. The T. matsutake genome sequence provides insight into the causes and consequences of genome size inflation.

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<![CDATA[Analysis of Epichloë festucae small secreted proteins in the interaction with Lolium perenne]]> https://www.researchpad.co/article/5c6dc9b2d5eed0c48452a041

Epichloë festucae is an endophyte of the agriculturally important perennial ryegrass. This species systemically colonises the aerial tissues of this host where its growth is tightly regulated thereby maintaining a mutualistic symbiotic interaction. Recent studies have suggested that small secreted proteins, termed effectors, play a vital role in the suppression of host defence responses. To date only a few effectors with important roles in mutualistic interactions have been described. Here we make use of the fully assembled E. festucae genome and EffectorP to generate a suite of 141 effector candidates. These were analysed with respect to their genome location and expression profiles in planta and in several symbiosis-defective mutants. We found an association between effector candidates and a class of transposable elements known as MITEs, but no correlation with other dynamic features of the E. festucae genome, such as transposable element-rich regions. Three effector candidates and a small GPI-anchored protein were chosen for functional analysis based on their high expression in planta compared to in culture and their differential regulation in symbiosis defective E. festucae mutants. All three candidate effector proteins were shown to possess a functional signal peptide and two could be detected in the extracellular medium by western blotting. Localization of the effector candidates in planta suggests that they are not translocated into the plant cell, but rather, are localized in the apoplastic space or are attached to the cell wall. Deletion and overexpression of the effector candidates, as well as the putative GPI-anchored protein, did not affect the plant growth phenotype or restrict growth of E. festucae mutants in planta. These results indicate that these proteins are either not required for the interaction at the observed life stages or that there is redundancy between effectors expressed by E. festucae.

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<![CDATA[Apollo: Democratizing genome annotation]]> https://www.researchpad.co/article/5c648d41d5eed0c484c823a0

Genome annotation is the process of identifying the location and function of a genome's encoded features. Improving the biological accuracy of annotation is a complex and iterative process requiring researchers to review and incorporate multiple sources of information such as transcriptome alignments, predictive models based on sequence profiles, and comparisons to features found in related organisms. Because rapidly decreasing costs are enabling an ever-growing number of scientists to incorporate sequencing as a routine laboratory technique, there is widespread demand for tools that can assist in the deliberative analytical review of genomic information. To this end, we present Apollo, an open source software package that enables researchers to efficiently inspect and refine the precise structure and role of genomic features in a graphical browser-based platform. Some of Apollo’s newer user interface features include support for real-time collaboration, allowing distributed users to simultaneously edit the same encoded features while also instantly seeing the updates made by other researchers on the same region in a manner similar to Google Docs. Its technical architecture enables Apollo to be integrated into multiple existing genomic analysis pipelines and heterogeneous laboratory workflow platforms. Finally, we consider the implications that Apollo and related applications may have on how the results of genome research are published and made accessible.

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<![CDATA[Finding the needle in a haystack: Mapping antifungal drug resistance in fungal pathogen by genomic approaches]]> https://www.researchpad.co/article/5c5ca2fad5eed0c48441eee3 ]]> <![CDATA[Distribution of Scedosporium species in soil from areas with high human population density and tourist popularity in six geographic regions in Thailand]]> https://www.researchpad.co/article/5c521862d5eed0c484797eef

Scedosporium is a genus comprising at least 10 species of airborne fungi (saprobes) that survive and grow on decaying organic matter. These fungi are found in high density in human-affected areas such as sewage-contaminated water, and five species, namely Scedosporium apiospermum, S. boydii, S. aurantiacum, S. dehoogii, and S. minutisporum, cause human infections. Thailand is a popular travel destination in the world, with many attractions present in densely populated areas; thus, large numbers of people may be exposed to pathogens present in these areas. We conducted a comprehensive survey of Scedosporium species in 350 soil samples obtained from 35 sites of high human population density and tourist popularity distributed over 23 provinces and six geographic regions of Thailand. Soil suspensions of each sample were inoculated on three plates of Scedo-Select III medium to isolate Scedosporium species. In total, 191 Scedosporium colonies were isolated from four provinces. The species were then identified using PCR and sequencing of the beta-tubulin (BT2) gene. Of the 191 isolates, 188 were S. apiospermum, one was S. dehoogii, and species of two could not be exactly identified. Genetic diversity analysis revealed high haplotype diversity of S. apiospermum. Soil is a major ecological niche for Scedosporium and may contain S. apiospermum populations with high genetic diversity. This study of Scedosporium distribution might encourage health care providers to consider Scedosporium infection in their patients.

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<![CDATA[Danger signals activate a putative innate immune system during regeneration in a filamentous fungus]]> https://www.researchpad.co/article/5c0ae430d5eed0c4845891aa

The ability to respond to injury is a biological process shared by organisms of different kingdoms that can even result in complete regeneration of a part or structure that was lost. Due to their immobility, multicellular fungi are prey to various predators and are therefore constantly exposed to mechanical damage. Nevertheless, our current knowledge of how fungi respond to injury is scarce. Here we show that activation of injury responses and hyphal regeneration in the filamentous fungus Trichoderma atroviride relies on the detection of two danger or alarm signals. As an early response to injury, we detected a transient increase in cytosolic free calcium ([Ca2+]c) that was promoted by extracellular ATP, and which is likely regulated by a mechanism of calcium-induced calcium-release. In addition, we demonstrate that the mitogen activated protein kinase Tmk1 plays a key role in hyphal regeneration. Calcium- and Tmk1-mediated signaling cascades activated major transcriptional changes early following injury, including induction of a set of regeneration associated genes related to cell signaling, stress responses, transcription regulation, ribosome biogenesis/translation, replication and DNA repair. Interestingly, we uncovered the activation of a putative fungal innate immune response, including the involvement of HET domain genes, known to participate in programmed cell death. Our work shows that fungi and animals share danger-signals, signaling cascades, and the activation of the expression of genes related to immunity after injury, which are likely the result of convergent evolution.

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<![CDATA[TPP riboswitch-dependent regulation of an ancient thiamin transporter in Candida]]> https://www.researchpad.co/article/5b28b39a463d7e126303d2ab

Riboswitches are non-coding RNA molecules that regulate gene expression by binding to specific ligands. They are primarily found in bacteria. However, one riboswitch type, the thiamin pyrophosphate (TPP) riboswitch, has also been described in some plants, marine protists and fungi. We find that riboswitches are widespread in the budding yeasts (Saccharomycotina), and they are most common in homologs of DUR31, originally described as a spermidine transporter. We show that DUR31 (an ortholog of N. crassa gene NCU01977) encodes a thiamin transporter in Candida species. Using an RFP/riboswitch expression system, we show that the functional elements of the riboswitch are contained within the native intron of DUR31 from Candida parapsilosis, and that the riboswitch regulates splicing in a thiamin-dependent manner when RFP is constitutively expressed. The DUR31 gene has been lost from Saccharomyces, and may have been displaced by an alternative thiamin transporter. TPP riboswitches are also present in other putative transporters in yeasts and filamentous fungi. However, they are rare in thiamin biosynthesis genes THI4 and THI5 in the Saccharomycotina, and have been lost from all genes in the sequenced species in the family Saccharomycetaceae, including S. cerevisiae.

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<![CDATA[Functional Diversification of Hsp40: Distinct J-Protein Functional Requirements for Two Prions Allow for Chaperone-Dependent Prion Selection]]> https://www.researchpad.co/article/5989db09ab0ee8fa60bc991f

Yeast prions are heritable amyloid aggregates of functional yeast proteins; their propagation to subsequent cell generations is dependent upon fragmentation of prion protein aggregates by molecular chaperone proteins. Mounting evidence indicates the J-protein Sis1 may act as an amyloid specificity factor, recognizing prion and other amyloid aggregates and enabling Ssa and Hsp104 to act in prion fragmentation. Chaperone interactions with prions, however, can be affected by variations in amyloid-core structure resulting in distinct prion variants or ‘strains’. Our genetic analysis revealed that Sis1 domain requirements by distinct variants of [PSI+] are strongly dependent upon overall variant stability. Notably, multiple strong [PSI+] variants can be maintained by a minimal construct of Sis1 consisting of only the J-domain and glycine/phenylalanine-rich (G/F) region that was previously shown to be sufficient for cell viability and [RNQ+] prion propagation. In contrast, weak [PSI+] variants are lost under the same conditions but maintained by the expression of an Sis1 construct that lacks only the G/F region and cannot support [RNQ+] propagation, revealing mutually exclusive requirements for Sis1 function between these two prions. Prion loss is not due to [PSI+]-dependent toxicity or dependent upon a particular yeast genetic background. These observations necessitate that Sis1 must have at least two distinct functional roles that individual prions differentially require for propagation and which are localized to the glycine-rich domains of the Sis1. Based on these distinctions, Sis1 plasmid-shuffling in a [PSI+]/[RNQ+] strain permitted J-protein-dependent prion selection for either prion. We also found that, despite an initial report to the contrary, the human homolog of Sis1, Hdj1, is capable of [PSI+] prion propagation in place of Sis1. This conservation of function is also prion-variant dependent, indicating that only one of the two Sis1-prion functions may have been maintained in eukaryotic chaperone evolution.

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<![CDATA[Comparative Genomics of Plant Fungal Pathogens: The Ustilago-Sporisorium Paradigm]]> https://www.researchpad.co/article/5989da36ab0ee8fa60b86322 ]]> <![CDATA[Utilization of a Conidia-Deficient Mutant to Study Sexual Development in Fusarium graminearum]]> https://www.researchpad.co/article/5989d9f6ab0ee8fa60b7021f

Transcriptome analysis is a widely used approach to study the molecular mechanisms underlying development and the responses of fungi to environmental cues. However, it is difficult to obtain cells with a homogeneous status from the sexually-induced culture of the plant pathogenic fungus Fusarium graminearum. In this study, we provided phenotypic and genetic evidence to show that the current conditions applied for perithecia induction inevitably highly induced asexual sporulation in this fungus. We also found that hundreds of genes under the control of the conidiation-specific gene ABAA were unnecessarily upregulated after perithecia induction. Deletion of ABAA specifically blocked conidia production in both the wild-type strain and sexually-defective mutants during sexual development. Taken together, our results suggest that the abaA strain could be used as a background strain for studies of the initial stages of perithecia production in F. graminearum. Further comparative transcriptome analysis between the abaA mutant and the sexually-defective transcription factor mutant carrying the ABAA deletion would contribute to the construction of the genetic networks involved in perithecia development in F. graminearum.

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<![CDATA[Insights into the Utility of the Focal Adhesion Scaffolding Proteins in the Anaerobic Fungus Orpinomyces sp. C1A]]> https://www.researchpad.co/article/5989dad3ab0ee8fa60bb7154

Focal adhesions (FAs) are large eukaryotic multiprotein complexes that are present in all metazoan cells and function as stable sites of tight adhesion between the extracellular matrix (ECM) and the cell’s cytoskeleton. FAs consist of anchor membrane protein (integrins), scaffolding proteins (e.g. α-actinin, talin, paxillin, and vinculin), signaling proteins of the IPP complex (e.g. integrin-linked kinase, α-parvin, and PINCH), and signaling kinases (e.g. focal adhesion kinase (FAK) and Src kinase). While genes encoding complete focal adhesion machineries are present in genomes of all multicellular Metazoa; incomplete machineries were identified in the genomes of multiple non-metazoan unicellular Holozoa, basal fungal lineages, and amoebozoan representatives. Since a complete FA machinery is required for functioning, the putative role, if any, of these incomplete FA machineries is currently unclear. We sought to examine the expression patterns of FA-associated genes in the anaerobic basal fungal isolate Orpinomyces sp. strain C1A under different growth conditions and at different developmental stages. Strain C1A lacks clear homologues of integrin, and the two signaling kinases FAK and Src, but encodes for all scaffolding proteins, and the IPP complex proteins. We developed a protocol for synchronizing growth of C1A cultures, allowing for the collection and mRNA extraction from flagellated spores, encysted germinating spores, active zoosporangia, and late inactive sporangia of strain C1A. We demonstrate that the genes encoding the FA scaffolding proteins α-actinin, talin, paxillin, and vinculin are indeed transcribed under all growth conditions, and at all developmental stages of growth. Further, analysis of the observed transcriptional patterns suggests the putative involvement of these components in alternative non-adhesion-specific functions, such as hyphal tip growth during germination and flagellar assembly during zoosporogenesis. Based on these results, we propose putative alternative functions for such proteins in the anaerobic gut fungi. Our results highlight the presumed diverse functionalities of FA scaffolding proteins in basal fungi.

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<![CDATA[A Small Protein Associated with Fungal Energy Metabolism Affects the Virulence of Cryptococcus neoformans in Mammals]]> https://www.researchpad.co/article/5989da08ab0ee8fa60b76738

The pathogenic yeast Cryptococcus neoformans causes cryptococcosis, a life-threatening fungal disease. C. neoformans has multiple virulence mechanisms that are non-host specific, induce damage and interfere with immune clearance. Microarray analysis of C. neoformans strains serially passaged in mice associated a small gene (CNAG_02591) with virulence. This gene, hereafter identified as HVA1 (hypervirulence-associated protein 1), encodes a protein that has homologs of unknown function in plant and animal fungi, consistent with a conserved mechanism. Expression of HVA1 was negatively correlated with virulence and was reduced in vitro and in vivo in both mouse- and Galleria-passaged strains of C. neoformans. Phenotypic analysis in hva1Δ and hva1Δ+HVA1 strains revealed no significant differences in established virulence factors. Mice infected intravenously with the hva1Δ strain had higher fungal burden in the spleen and brain, but lower fungal burden in the lungs, and died faster than mice infected with H99W or the hva1Δ+HVA1 strain. Metabolomics analysis demonstrated a general increase in all amino acids measured in the disrupted strain and a block in the TCA cycle at isocitrate dehydrogenase, possibly due to alterations in the nicotinamide cofactor pool. Macrophage fungal burden experiments recapitulated the mouse hypervirulent phenotype of the hva1Δ strain only in the presence of exogenous NADPH. The crystal structure of the Hva1 protein was solved, and a comparison of structurally similar proteins correlated with the metabolomics data and potential interactions with NADPH. We report a new gene that modulates virulence through a mechanism associated with changes in fungal metabolism.

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<![CDATA[Identification of Degenerate Nuclei and Development of a SCAR Marker for Flammulina velutipes]]> https://www.researchpad.co/article/5989d9ecab0ee8fa60b6cdb2

Flammulina velutipes is one of the major edible mushrooms in the world. Recently, abnormalities that have a negative impact on crop production have been reported in this mushroom. These symptoms include slow vegetative growth, a compact mycelial mat, and few or even no fruiting bodies. The morphologies and fruiting capabilities of monokaryons of wild-type and degenerate strains that arose through arthrospore formation were investigated through test crossing. Only one monokaryotic group of the degenerate strains and its hybrid strains showed abnormal phenotypes. Because the monokaryotic arthrospore has the same nucleus as the parent strain, these results indicated that only one aberrant nucleus of the two nuclei in the degenerate strain was responsible for the degeneracy. A sequence-characterized amplified region marker that is linked to the degenerate monokaryon was identified based on a polymorphic sequence that was generated using random primers. Comparative analyses revealed the presence of a degenerate-specific genomic region in a telomere, which arose via the transfer of a genomic fragment harboring a putative helicase gene. Our findings have narrowed down the potential molecular targets responsible for this phenotype for future studies and have provided a marker for the detection of degenerate strains.

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<![CDATA[Signature Gene Expression Reveals Novel Clues to the Molecular Mechanisms of Dimorphic Transition in Penicillium marneffei]]> https://www.researchpad.co/article/5989da11ab0ee8fa60b79826

Systemic dimorphic fungi cause more than one million new infections each year, ranking them among the significant public health challenges currently encountered. Penicillium marneffei is a systemic dimorphic fungus endemic to Southeast Asia. The temperature-dependent dimorphic phase transition between mycelium and yeast is considered crucial for the pathogenicity and transmission of P. marneffei, but the underlying mechanisms are still poorly understood. Here, we re-sequenced P. marneffei strain PM1 using multiple sequencing platforms and assembled the genome using hybrid genome assembly. We determined gene expression levels using RNA sequencing at the mycelial and yeast phases of P. marneffei, as well as during phase transition. We classified 2,718 genes with variable expression across conditions into 14 distinct groups, each marked by a signature expression pattern implicated at a certain stage in the dimorphic life cycle. Genes with the same expression patterns tend to be clustered together on the genome, suggesting orchestrated regulations of the transcriptional activities of neighboring genes. Using qRT-PCR, we validated expression levels of all genes in one of clusters highly expressed during the yeast-to-mycelium transition. These included madsA, a gene encoding MADS-box transcription factor whose gene family is exclusively expanded in P. marneffei. Over-expression of madsA drove P. marneffei to undergo mycelial growth at 37°C, a condition that restricts the wild-type in the yeast phase. Furthermore, analyses of signature expression patterns suggested diverse roles of secreted proteins at different developmental stages and the potential importance of non-coding RNAs in mycelium-to-yeast transition. We also showed that RNA structural transition in response to temperature changes may be related to the control of thermal dimorphism. Together, our findings have revealed multiple molecular mechanisms that may underlie the dimorphic transition in P. marneffei, providing a powerful foundation for identifying molecular targets for mechanism-based interventions.

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<![CDATA[Seasonal Succession of Fungi Associated with Ips typographus Beetles and Their Phoretic Mites in an Outbreak Region of Finland]]> https://www.researchpad.co/article/5989da72ab0ee8fa60b95474

The ophiostomatoid fungi (Microascales and Ophiostomatales, Ascomycota) are common associates of Ips typographus, and include tree pathogens and species responsible for blue-stain of timber. Fungal assemblages associated with I. typographus have varied considerably between studies but few investigations have attempted to explain this variation. For this reason, we assessed the overall cultivable fungal diversity associated with I. typographus in a storm-felled spruce forest in south-eastern Finland. Fungi were isolated from the individually collected beetles as well as their phoretic mites in spring, summer and autumn, including different life stages of the beetle (hibernation, dispersal flight and first generation). The internal transcribed spacer (ITS) gene region was used to identify the fungi. A total of 32 operational taxonomic units (OTUs) were found and these resided in four fungal phyla/subphyla (24 Ascomycota, 2 Basidiomycota, 5 Mucoromycotina, 1 Mortierellomycotina) in association with adult bark beetles. Ophiostomatoid species were the most commonly detected fungal associates. A generalized linear model analysis showed a clear association between fungal communities and season, indicating seasonal succession among I. typographus-associated fungi. The season of sampling appears to be an important factor that has resulted in inconsistencies between results in previous studies. Many of these fungi were also found on phoretic mites and their presence or absence could have influenced variation in patterns of association.

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<![CDATA[Genome-Wide Identification and Comprehensive Analyses of the Kinomes in Four Pathogenic Microsporidia Species]]> https://www.researchpad.co/article/5989da3cab0ee8fa60b88540

Microsporidia have attracted considerable attention because they infect a wide range of hosts, from invertebrates to vertebrates, and cause serious human diseases and major economic losses in the livestock industry. There are no prospective drugs to counteract this pathogen. Eukaryotic protein kinases (ePKs) play a central role in regulating many essential cellular processes and are therefore potential drug targets. In this study, a comprehensive summary and comparative analysis of the protein kinases in four microsporidia–Enterocytozoon bieneusi, Encephalitozoon cuniculi, Nosema bombycis and Nosema ceranae–was performed. The results show that there are 34 ePKs and 4 atypical protein kinases (aPKs) in E. bieneusi, 29 ePKs and 6 aPKs in E. cuniculi, 41 ePKs and 5 aPKs in N. bombycis, and 27 ePKs and 4 aPKs in N. ceranae. These data support the previous conclusion that the microsporidian kinome is the smallest eukaryotic kinome. Microsporidian kinomes contain only serine-threonine kinases and do not contain receptor-like and tyrosine kinases. Many of the kinases related to nutrient and energy signaling and the stress response have been lost in microsporidian kinomes. However, cell cycle-, development- and growth-related kinases, which are important to parasites, are well conserved. This reduction of the microsporidian kinome is in good agreement with genome compaction, but kinome density is negatively correlated with proteome size. Furthermore, the protein kinases in each microsporidian genome are under strong purifying selection pressure. No remarkable differences in kinase family classification, domain features, gain and/or loss, and selective pressure were observed in these four species. Although microsporidia adapt to different host types, the coevolution of microsporidia and their hosts was not clearly reflected in the protein kinases. Overall, this study enriches and updates the microsporidian protein kinase database and may provide valuable information and candidate targets for the design of treatments for pathogenic diseases.

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<![CDATA[Microbial Potential for Ecosystem N Loss Is Increased by Experimental N Deposition]]> https://www.researchpad.co/article/5989da36ab0ee8fa60b86591

Fossil fuel combustion and fertilizer use has increased the amount of biologically available N entering terrestrial ecosystems. Nonetheless, our understanding of how anthropogenic N may alter the physiological mechanisms by which soil microorganisms cycle N in soil is still developing. Here, we applied shotgun metagenomics to a replicated long-term field experiment to determine how two decades of experimental N deposition, at a rate expected by mid-century, has affected the genetic potential of the soil microbial community to cycle N in soils. Experimental N deposition lead to a significant and persistent increase in functional assemblages mediating N cycle transformations associated with ecosystem N loss (i.e., denitrification and nitrification), whereas functional assemblages associated with N input and retention (i.e., N fixation and microbial N assimilation) were less positively affected. Furthermore, the abundance and composition of microbial taxa, as well as functional assemblages involved in housekeeping functions (i.e., DNA replication) were unaffected by experimental N deposition. Taken together, our results suggest that functional genes and gene pathways associated with ecosystem N loss have been favored by experimental N deposition, which may represent a genetic mechanism fostering increased N loss as anthropogenic N deposition increases in the future.

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<![CDATA[Characterization of Five Novel Mitoviruses in the White Pine Blister Rust Fungus Cronartium ribicola]]> https://www.researchpad.co/article/5989d9e5ab0ee8fa60b6aec6

The white pine blister rust (WPBR) fungus Cronartium ribicola (J.C. Fisch.) is an exotic invasive forest pathogen causing severe stem canker disease of native white pine trees (subgenus Strobus) in North America. The present study reports discovery of five novel mitoviruses in C. ribicola by deep RNA sequencing. The complete genome of each mitovirus was determined by rapid amplification of cDNA ends (RACE) and reverse transcriptase-polymerase chain reaction (RT-PCR). A single open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) was detected in each of the viral genomes using mitochondrial genetic codes. Phylogenetic analysis indicated that the C. ribicola mitoviruses (CrMV1 to CrMV5) are new putative species of the genus Mitovirus. qRT-PCR and RNA-Seq analyses revealed that viral RNAs were significantly increased in fungal mycelia in cankered pine stems compared to expression during two different stages of spore development, suggesting that viral genome replication and transcription benefit from active growth of the host fungus. CrMVs were widespread with relatively high levels of minor allele frequency (MAF) in western North America. As the first report of mitoviruses in the Class Pucciniomycetes, this work allows further investigation of the dynamics of a viral community in the WPBR pathosystem, including potential impacts that may affect pathogenicity and virulence of the host fungus.

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